ABSTRACT: Tubular urethra formation in humans was described as distal-opening-proximal-closing ?zippers?, but similar process has never been shown in any published animal models, including mice and rats. Thus, the significance of this work stems from the knowledge gap in developmental and cellular mechanism of distal- opening-proximal-closing in tubular urethra formation. Recently we studied the embryology and sexual differentiation of the guinea pig external genitalia, and revealed the penile masculinization process is similar to humans. Canalization and dorsal-to-ventral movement of the urethral canal lead to the urethral groove formation in both sexes, and then the urethral groove of males performs distal-opening-proximal-closing to form tubular urethra. In addition, more nuclear localized androgen receptors were found in proximal portion of genital tubercles of males than females during tubular urethra formation. Antiandrogen treatment at specific time window of embryonic development caused hypospadias, methyltestosterone administration at same time could induce penis formation in females. Our preliminary data suggest that cell proliferation and cell death between dorsal and ventral aspects of genital tubercle may lead to urethral groove formation. This process starts from proximal region of genital tubercle and extends up to distal glans. The urethral closing starts in the proximal region in males at E29, but the distal opening process continues until the extension of urethral groove reaches up to and opens the entire glans at E31, thus the distal opening and proximal closing process can be observed in the same tubercle. Based on these recent studies in our lab, we propose this project to determine the developmental and cellular mechanism of distal opening and proximal closing in urethral tube formation. The project is based on a central hypothesis that differential cell proliferation and cell death determine distal-opening-proximal-closing in urethral tube formation during guinea pig penile development. The first aim is to determine the cellular process of urethral groove formation using guinea pig model. This will be conducted by examine the differential proliferation, death, size and shape of urethral epithelial and adjacent mesenchymal cells between dorsal and ventral aspects in developing genital tubercle. Our second aim is to assess the mechanism of how androgen initiates proximal urethral tube formation in guinea pig penile masculinization while the distal is still opening. This will be conducted by first examining the variation in cell proliferation, cell death, cell size and cell shape in both mesenchyme and epithelia between proximal and distal portions of genital tubercles and among control males, control females, anti-androgen treated males and androgen treated females; and then detecting changes in developmental gene expression between proximal and distal of genital tubercle and among these four groups in genome level. The findings from this work will lay groundwork for further elucidation of the underlying molecular mechanisms of distal opening and proximal closing during tubular urethra formation. The expected outcomes will form a basis for exploration of the etiology and detailed mechanism of hypospadias in humans.